what are the methods available for protein extraction from cell line? - (Jul/18/2008 )

Hi, I would like to extract transmembrane protein/ enzymes from the cells. Which lysis method is commonly used? Usually how many cells needed for protein extraction if I need abt 100-500ug total protein been extracted from the cells?

I plan to load the cells in culture plate, which type of culture plate should I used? 24-wells? 12-wells? Or 6-well plate?

-sasoriza-

QUOTE (sasoriza @ Jul 18 2008, 09:36 AM)

Hi, I would like to extract transmembrane protein/ enzymes from the cells. Which lysis method is commonly used? Usually how many cells needed for protein extraction if I need abt 100-500ug total protein been extracted from the cells?

I plan to load the cells in culture plate, which type of culture plate should I used? 24-wells? 12-wells? Or 6-well plate?

Hi! The lysis method typically depends on cellular type. We typically use RIPA buffer (150mM NaCL, 1% NP-40, 0.5% SDS, 50mM TrispH8, 2mM EDTA) or ELB-softer (150mM NaCl, 50mM Hepes pH7, 5mM EDTA, 0,1% NP-40), both of them plus protease inhibitors added prior to use. Typically for protein I use 6 wp or 6cm dishes (4-8x10^6 cells). I performed a previous PBS wash, then lysis in 200ul and 3 cycles of somication for 5 min; finally a centrifug at max speed for 5 min to pellet debris. The SN contains de protein. However, there are lots of methods, a freezing-cooling method from dry ice to 37ºC, or rotation at 4ºC, etc...

-Estersan-

QUOTE (Estersan @ Jul 18 2008, 07:46 AM)

QUOTE (sasoriza @ Jul 18 2008, 09:36 AM)

Hi, I would like to extract transmembrane protein/ enzymes from the cells. Which lysis method is commonly used? Usually how many cells needed for protein extraction if I need abt 100-500ug total protein been extracted from the cells?

I plan to load the cells in culture plate, which type of culture plate should I used? 24-wells? 12-wells? Or 6-well plate?

Hi! The lysis method typically depends on cellular type. We typically use RIPA buffer (150mM NaCL, 1% NP-40, 0.5% SDS, 50mM TrispH8, 2mM EDTA) or ELB-softer (150mM NaCl, 50mM Hepes pH7, 5mM EDTA, 0,1% NP-40), both of them plus protease inhibitors added prior to use. Typically for protein I use 6 wp or 6cm dishes (4-8x10^6 cells). I performed a previous PBS wash, then lysis in 200ul and 3 cycles of somication for 5 min; finally a centrifug at max speed for 5 min to pellet debris. The SN contains de protein. However, there are lots of methods, a freezing-cooling method from dry ice to 37ºC, or rotation at 4ºC, etc...

What about if using protein extraction kit? Will it be more efficient and more easy to get more protein from the cells?

-sasoriza-

Personally I won't spend mony on protein extraction kits because you can easily make up lysis buffer for a fraction of the cost. RIPA is a good buffer if you just want to extract as much as you can then western/IP. However, if you want to do enzyme assays, you may need to use non-detergent buffer. What Estersan suggest is a very good starting point. You will need to work out for your own proteins depending on their properties.

-Michelle4-

QUOTE (Michelle4 @ Jul 21 2008, 02:31 AM)

Personally I won't spend mony on protein extraction kits because you can easily make up lysis buffer for a fraction of the cost. RIPA is a good buffer if you just want to extract as much as you can then western/IP. However, if you want to do enzyme assays, you may need to use non-detergent buffer. What Estersan suggest is a very good starting point. You will need to work out for your own proteins depending on their properties.

Actually what I plan to do is to extract the total proteins from the cells, then use for enzyme assay. The enzyme of interest is cellular tyrosinase enzyme. Can I use 0.1M sodium phosphate buffer (pH 6.8) containing 1% Triton-X with protease inhibitor added into the lysis buffer to lyse the cells? I am new and never do protein extraction before....can somebody pls provide me some relavant protocol on cell lysis and protein extraction for enzymetic study?Thank you.....

-sasoriza-

QUOTE (sasoriza @ Jul 21 2008, 05:48 PM)

QUOTE (Michelle4 @ Jul 21 2008, 02:31 AM)

Personally I won't spend mony on protein extraction kits because you can easily make up lysis buffer for a fraction of the cost. RIPA is a good buffer if you just want to extract as much as you can then western/IP. However, if you want to do enzyme assays, you may need to use non-detergent buffer. What Estersan suggest is a very good starting point. You will need to work out for your own proteins depending on their properties.

Actually what I plan to do is to extract the total proteins from the cells, then use for enzyme assay. The enzyme of interest is cellular tyrosinase enzyme. Can I use 0.1M sodium phosphate buffer (pH 6.8) containing 1% Triton-X with protease inhibitor added into the lysis buffer to lyse the cells? I am new and never do protein extraction before....can somebody pls provide me some relavant protocol on cell lysis and protein extraction for enzymetic study?Thank you.....

Actualy i think you are going the right way....to isolate transmembrane proteins you have to use a non-ionic detergent such as Triton-X and not SDS....but the standard lysis buffer for membrane protein isolation cannot be used in this case because you need to supplement it with protease inhibitors which are not suitable for enzyme assays. I donot have a protocol on this at hand but will surely post it once i find it.

-laila-

QUOTE (laila @ Sep 26 2008, 04:04 AM)

QUOTE (sasoriza @ Jul 21 2008, 05:48 PM)

QUOTE (Michelle4 @ Jul 21 2008, 02:31 AM)

Personally I won't spend mony on protein extraction kits because you can easily make up lysis buffer for a fraction of the cost. RIPA is a good buffer if you just want to extract as much as you can then western/IP. However, if you want to do enzyme assays, you may need to use non-detergent buffer. What Estersan suggest is a very good starting point. You will need to work out for your own proteins depending on their properties.

Actually what I plan to do is to extract the total proteins from the cells, then use for enzyme assay. The enzyme of interest is cellular tyrosinase enzyme. Can I use 0.1M sodium phosphate buffer (pH 6.8) containing 1% Triton-X with protease inhibitor added into the lysis buffer to lyse the cells? I am new and never do protein extraction before....can somebody pls provide me some relavant protocol on cell lysis and protein extraction for enzymetic study?Thank you.....

Actualy i think you are going the right way....to isolate transmembrane proteins you have to use a non-ionic detergent such as Triton-X and not SDS....but the standard lysis buffer for membrane protein isolation cannot be used in this case because you need to supplement it with protease inhibitors which are not suitable for enzyme assays. I donot have a protocol on this at hand but will surely post it once i find it.

Thanks for your reply. My friend ask me to try M-Per mammalian protein lysis reagent from Pierce. And I have tried this lysis reagent to lyse my cells. Then I collect the supernatant and add in the substract for the enzymatic reaction. Color formed after incubation at 37 degree celcius. So does it mean I can use this lysis reagent to extract the tyrosinase enzyme from my cells and use for the enzyme assay?I found that M-Per mamalian protein lysis reagent is very convenient to use, just incubate at room temparature for 5 min and spin down to get the supernatant. No need freeze-thaw, sonicate.....etc, and save time.